Evaluation of an Electricity-free,Culture-based Approach for Detecting Typhoidal Salmonella Bacteremia during Enteric Fever in a High Burden,Resource-limited Setting

Background

In many rural areas at risk for enteric fever, there are few data on Salmonella enterica serotypes Typhi (S. Typhi) and Paratyphi (S. Paratyphi) incidence, due to limited laboratory capacity for microbiologic culture. Here, we describe an approach that permits recovery of the causative agents of enteric fever in such settings. This approach involves the use of an electricity-free incubator based upon use of phase-change materials. We compared this against conventional blood culture for detection of typhoidal Salmonella.

Methodology/Principal Findings

Three hundred and four patients with undifferentiated fever attending the outpatient and emergency departments of a public hospital in the Kathmandu Valley of Nepal were recruited. Conventional blood culture was compared against an electricity-free culture approach. Blood from 66 (21.7%) patients tested positive for a Gram-negative bacterium by at least one of the two methods. Sixty-five (21.4%) patients tested blood culture positive for S. Typhi (30; 9.9%) or S. Paratyphi A (35; 11.5%). From the 65 individuals with culture-confirmed enteric fever, 55 (84.6%) were identified by the conventional blood culture and 60 (92.3%) were identified by the experimental method. Median time-to-positivity was 2 days for both procedures. The experimental approach was falsely positive due to probable skin contaminants in 2 of 239 individuals (0.8%). The percentages of positive and negative agreement for diagnosis of enteric fever were 90.9% (95% CI: 80.0%–97.0%) and 96.0% (92.7%–98.1%), respectively. After initial incubation, Salmonella isolates could be readily recovered from blood culture bottles maintained at room temperature for six months.

Conclusions/Significance

A simple culture approach based upon a phase-change incubator can be used to isolate agents of enteric fever. This approach could be used as a surveillance tool to assess incidence and drug resistance of the etiologic agents of enteric fever in settings without reliable local access to electricity or local diagnostic microbiology laboratories.     

Computational Study of Plasmon Interaction in Organic Media: a Comparison Between Analytical and Numerical Model for Dimer

Plasmonics - In the present work, an investigation of wavelength-dependent absorption spectrum of spherical nanoparticles with different arrangements in organic medium using discrete dipole...     

Histopathology of Mycotoxicosis produced in Swiss albino mice by metabolites of some fungal isolates.

Of 199 fungal cultures isolated from some of the common cereals collected from different parts of Uttar Pradesh and Madhya Pradesh, 70 produced toxic metabolites. Of the 70 fungi isolated, 59 produced toxins which caused visible lesions in livers, kidneys, and spleens but did not cause mortality. Toxicity, graded in terms of the mortality rate and the extent of lesions in livers, kidneys, and spleens, was found to be highest in various species of Aspergillus (40%), followed by Chaetomium spp. (31%).     

Cytogenetic identification of interspecies cell-line contamination: procedures for non-cytogeneticists

A few simple procedures and cytological characteristics are described for identification of cell-line contamination involving human cells, or cells of several species of common laboratory and domestic animals. These include gross chromosome morphology, C-banding, fluorescence, and the nucleolus organizer regions as revealed by silver straining.     

Endosulfan-induced apoptosis and glutathione depletion in human peripheral blood mononuclear cells: Attenuation by N-acetylcysteine

Present study investigated whether endosulfan, an organochlorine pesticide is able to deplete glutathione (GSH) and induce apoptosis in human peripheral blood mononuclear cells (PBMC) in vitro. The role of oxidative stress in the induction of apoptosis was also evaluated by the measurement of the GSH level in cell lysate. The protective role of N-acetylcysteine (NAC) on endosulfan-induced apoptosis was also studied. Isolated human PBMC were exposed to increasing concentrations (0-100 microM) of endosulfan (alpha/beta at 70:30 mixture) alone and in combination with NAC (20 microM) up to 24 h. Apoptotic cell death was determined by Annexin-V Cy3.18 binding and DNA fragmentation assays. Cellular GSH level was measured using dithionitrobenzene. Endosulfan at low concentrations, i.e., 5 and 10 microM, did not cause significant death during 6 h/12 h incubation, whereas a concentration-dependent cell death was observed at 24 h. DNA fragmentation analysis revealed no appreciable difference between control cells and 5 microM/10 microM endosulfan treated cells, where only high molecular weight DNA band was observed. Significant ladder formation was observed at higher concentration, which is indicative of apoptotic cell death. Intracellular GSH levels decreased significantly in endosulfan-treated cells in a dose-dependent manner, showing a close correlation between oxidative stress and degree of apoptosis of PBMC. Cotreatment with NAC attenuated GSH depletion as well as apoptosis. Our results provide experimental evidence of involvement of oxidative stress in endosulfan-mediated apoptosis in human PBMC in vitro.     

Experimental aspergillosis in the German cockroach Blattella germanica: a histopathological study

Inoculation of a spore suspension of Aspergillus flavus in the haemocoel of the German cockroach (Blatella germanica) resulted in large-scale invasion of various internal organs as seen in tissue sections stained with a fungal stain. The organs affected were the alimentary canal, fat bodies, muscle fibres, malpighian tubules and the cerebral neurosecretory cells. No invasion of the trachea and tracheoles was noted. This revised version was published online in June 2006 with corrections to the Cover Date.     

A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.     

Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.Poliovirus (PV) is the most extensively studied member of the picornavirus family and serves as a paradigm not only for picornaviruses but also for many of the nonretroviral positive strand RNA viruses (74). A schematic of the ∼7,500-nucleotide PV genome is shown in Fig. ​Fig.1A.1A. The 5′ end is linked covalently to a 22-amino-acid peptide termed VPg (virion protein genome linked) that is encoded by the 3B region of the genome. VPg and 3B are therefore used interchangeably. The 3′ end of the genome is terminated by a poly(rA) tail. Upon release of the genome into the host cell cytoplasm, genome translation is initiated by using the internal ribosome entry site. An ∼3,000-amino-acid polyprotein is produced. Complete cleavage of the polyprotein by virus-encoded proteases yields 10 proteins. The polyprotein can be divided further into three smaller polyproteins: P1, P2, and P3. P1 contains capsid proteins: VP0, VP3, and VP1. VP0 undergoes autocatalytic cleavage after genome encapsidation to produce VP4 and VP2 proteins. P2 performs host interaction functions required for robust virus multiplication, for example, shutoff of host cell translation and induction of vesicles employed for genome replication, the so-called replication complexes (RCs). P3 contains proteins that function most directly in genome replication, including the RNA-dependent RNA polymerase. Translation induces RCs, leading to genome replication. Early during infection, replicated genomes are employed as templates for translation, leading to an exponential amplification of RCs and replicated RNA. Ultimately, production of viral proteins ceases and replicated genomes are packaged. The use of RCs provides a barrier to genetic complementation; all proteins must be provided in cis, that is, produced from the RNA that they replicate.Open in a separate windowFIG. 1.PV genome organization and P3 processing pathway. (A) Schematic of the PV genome. The 5′ end of the genome is covalently linked to a peptide (VPg) encoded by the 3B region of the genome. The 3′ end contains a poly(rA) tail. Three cis-acting replication elements are known. oriL is located in 5′ NTR. oriR is located in the 3′ NTR. oriI is located in 2C-coding sequence for PV; the position of this element is virus dependent. oriI is the template for VPg uridylylation. Translation initiation employs an internal ribosome entry site (IRES). The single open reading frame encodes a polyprotein. P1 produces virion structural proteins as indicated. P2 produces proteins thought to participate in virus-host interactions required for genome replication. P3 produces proteins thought to participate directly in genome replication. Polyprotein processing is mediated by protease activity residing in 2A, 3C, and/or 3CD proteins. (B) Processing of the P3 precursor occurs by two independent pathways (60). There are major (I) and minor (II) pathways. In pathway I, processing between 3B and 3C yields 3AB and 3CD. In pathway II, processing between 3A and 3B yields 3A and 3BCD. 3BCD processing yields 3BC and 3D; 3BC processing yields 3B and 3C. Pathway II is proposed to function in genome replication and is not perturbed in the GG mutant.In addition to P3 proteins, genome replication requires three cis-acting replication elements (CREs): a cloverleaf structure located in the 5′ nontranslated region (NTR), termed oriL (left) (1, 5); a stem-loop structure located in 2C-coding sequence, termed oriI (internal) (30, 61); and a pseudoknot structure located in the 3′ NTR, termed oriR (right) (1, 40). The first step of genome replication is diuridylylation of VPg or a VPg-containing protein primer (62, 74). This reaction is templated by oriI but also requires oriL in a cell-free reaction and is catalyzed by the viral RNA-dependent RNA polymerase 3Dpol (4, 5, 11, 30, 61). In addition to the four terminal P3 cleavage products (3A, 3B, 3C, and 3D proteins) and the uncleaved P3 polyprotein, several “intermediates” are observed in infected cells (3AB, 3CD, and 3BCD proteins) (Fig. ​(Fig.1B)1B) (43, 57, 73). The major P3 cleavage pathway (I) produces 3AB and 3CD proteins; the minor P3 cleavage pathway (II) produces 3A and 3BCD proteins (Fig. ​(Fig.1B)1B) (60). In some cases, the intermediates have unique activities, specificities, and/or functions relative to their corresponding terminal cleavage products.Over the past 8 years much has been learned about oriI-templated VPg uridylylation in vitro for a variety of picornaviruses (28, 49, 53, 77, 92). However, it is still unclear whether or not VPg, 3BC(D), or 3AB is used in vivo to initiate genome replication. The VPg peptide can be uridylylated in vitro (62); however, VPg-pUpU does not chase efficiently into full-length RNA (81). 3BC(D) is uridylylated more efficiently than VPg in vitro, leading to the possibility that this precursor could be used in vivo (60). To date, 3AB has been uridylylated in vitro only in the presence of Mn²⁺ (66). In order to begin to probe the origin of VPg that is linked to picornaviral RNA, we created a PV mutant in which the cleavage site between 3B and 3C was changed from Gln-Gly to Gly-Gly (60). We refer to this mutant as GG. The GG mutation should be lethal for genome replication if use of the processed VPg peptide is absolutely required for genome replication. For the GG mutant, products of the major P3 cleavage pathway were no longer 3AB and 3CD but were now 3ABC and 3D instead. The kinetics of genome replication were reduced for the GG mutant relative to those for the wild type (WT). Surprisingly, the yield of replicated GG RNA was within an order of magnitude of that observed for WT RNA. Replicated GG RNA was then linked covalently to 3BC instead of VPg, as observed for WT PV. In spite of the substantial yield of replicated RNA, infectious virus was not recovered.We have performed a molecular characterization of the GG mutant. GG PV RNA is quasi-infectious. The low yield of virus recovery relative to replicated RNA reflects a block at a step in the PV multiplication cycle positioned after genome replication but prior to virus assembly. The existence of this step in the PV life cycle was suggested previously by Baltimore (8). Surprisingly, none of the defects associated with GG PV could be attributed to the absence of 3CD protease activity, suggesting that precursors larger than 3CD may be the primary proteases employed in vivo. All of the observed defects in GG PV multiplication were ameliorated in a pseudorevertant in which the 3B-3C cleavage site was changed from Gly-Gly to Glu-Gly. This mutant is referred to as EG. Molecular characterization of EG PV revealed for the first time a trans-complementable function for 3CD in genome replication. This observation supports a role for 3CD at a step preceding genome replication within RCs, perhaps RC formation. Our studies of EG PV confirmed the existence of a step between genome replication and virus assembly that requires 3CD and/or 3AB, thus providing compelling evidence for genome replication and genome encapsidation as distinct steps in the multiplication cycle. This study highlights the utility of polyprotein cleavage site mutants for evaluation of the viral multiplication cycle.     

Discovery of [7-(2,6-dichlorophenyl)-5-methylbenzo [1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]amine--a potent, orally active Src kinase inhibitor with anti-tumor activity in preclinical assays

We describe the identification of [7-(2,6-dichlorophenyl)-5-methylbenzo [1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]amine (3), a potent, orally active Src inhibitor with desirable PK properties, demonstrated activity in human tumor cell lines and in animal models of tumor growth.     

Immuno-epidemiology of chronic bacterial and helminth co-infections: observations from the field and evidence from the laboratory

Co-infections can alter the host immune responses and modify the intensity and dynamics of concurrent parasitic species. The extent of this effect depends on the properties of the system and the mechanisms of host-parasite and parasite-parasite interactions. We examined the immuno-epidemiology of a chronic co-infection to reveal the immune mediated relationships between two parasites colonising independent organs, and the within-host molecular processes influencing the dynamics of infection at the host population level. The respiratory bacterium, Bordetella bronchiseptica, and the gastrointestinal helminth, Graphidium strigosum, were studied in the European rabbit (Oryctolagus cuniculus), using long-term field data and a laboratory experiment. We found that 65% of the rabbit population was co-infected with the two parasites; prevalence and intensity of co-infection increased with rabbit age and exhibited a strong seasonal pattern with the lowest values recorded during host breeding (from April to July) and the highest in the winter months. Laboratory infections showed no significant immune-mediated effects of the helminth on bacterial intensity in the lower respiratory tract but a higher abundance was observed in the nasal cavity during the chronic phase of the infection, compared with single bacterial infections. In contrast, B. bronchiseptica enhanced helminth intensity and this was consistent throughout the 4-month trial. These patterns were associated with changes in the immune profiles between singly and co-infected individuals for both parasites. This study confirmed the general observation that co-infections alter the host immune responses but also highlighted the often ignored role of bacterial infection in helminth dynamics. Additionally, we showed that G. strigosum had contrasting effects on B. bronchiseptica colonising different parts of the respiratory tract. At the host population level our findings suggest that B. bronchiseptica facilitates G. strigosum infection, and re-infection with G. strigosum assists in maintaining bacterial infection in the upper respiratory tract and thus long-term persistence.